A cellular high-throughput screening approach for therapeutic trans-cleaving ribozymes and RNAi against arbitrary mRNA disease targets

Abstract

Major bottlenecks in development of therapeutic post transcriptional gene silencing (PTGS) agents (e.g. ribozymes, RNA interference, antisense) include the challenge of mapping rare accessible regions of the mRNA target that are open for annealing and cleavage, testing and optimization of agents in human cells to identify lead agents, testing for cellular toxicity, and preclinical evaluation in appropriate animal models of disease. Methods for rapid and reliable cellular testing of PTGS agents are needed to identify potent lead candidates for optimization. Our goal was to develop a means of rapid assessment of many RNA agents to identify a lead candidate for a given mRNA associated with a disease state. We developed a rapid human cell-based screening platform to test efficacy of hammerhead ribozyme (hhRz) or RNA interference (RNAi) constructs, using a model retinal degeneration target, human rod opsin (RHO) mRNA. The focus is on RNA Drug Discovery for diverse retinal degeneration targets. To validate the approach, candidate hhRzs were tested against NUH↓ cleavage sites (N=G,C,A,U; H=C,A,U) within the target mRNA of secreted alkaline phosphatase (SEAP), a model gene expression reporter, based upon in silico predictions of mRNA accessibility. HhRzs were embedded in a larger stable adenoviral VAI RNA scaffold for high cellular expression, cytoplasmic trafficking, and stability. Most hhRz expression plasmids exerted statistically significant knockdown of extracellular SEAP enzyme activity when readily assayed by a fluorescence enzyme assay intended for high throughput screening (HTS). Kinetics of PTGS knockdown of cellular targets is measureable in live cells with the SEAP reporter. The validated SEAP HTS platform was transposed to identify lead PTGS agents against a model hereditary retinal degeneration target, RHO mRNA. Two approaches were used to physically fuse the model retinal gene target mRNA to the SEAP reporter mRNA. The most expedient way to evaluate a large set of potential VAI-hhRz expression plasmids against diverse NUH↓ cleavage sites uses cultured human HEK293S cells stably expressing a dicistronic Target-IRES-SEAP target fusion mRNA. Broad utility of this rational RNA drug discovery approach is feasible for any ophthalmological disease-relevant mRNA targets and any disease mRNA targets in general. The approach will permit rank ordering of PTGS agents based on potency to identify a lead therapeutic compound for further optimization.

Systematic Screening, Rational Development, and Initial Optimization of Efficacious RNA Silencing Agents for Human Rod Opsin Therapeutics

“…The secondary structure of the full-length hRHO mRNA transcript was analyzed for local folding regions with highly probable and stable secondary structures with substantial (≥8 nt) single-stranded regions. We employed three contemporary algorithms (MFold, SFold, OligoWalk [OW])30–32 to predict accessible regions in human RHO mRNA in an established approach developed in this lab, multiparameter prediction of RNA accessibility (mppRNA) 14–17,28. A three-dimensional (3D) RNA structure over limited regions can be reliably estimated from a publicly available algorithm called RNA Composer33 (in the public domain, http://rnacomposer.cs.put.poznan.pl); this algorithm draws on published RNA structured elements from the Protein Data Base (repository also for RNA crystal structures).…”

“…We have shown that the first major challenge or bottleneck in PTGS development is to identify regions that are accessible 16. We recently reported on a high-throughput screening (HTS) methodologic approach to identify a lead candidate PTGS agent to an arbitrary mRNA target 17. Here, we describe the identification of a lead candidate agent to hRHO mRNA and our initial efforts toward rational optimization.…”

Molecular Therapies for Inherited Retinal Diseases—Current Standing, Opportunities and Challenges

“…HhRzs are small RNA molecules causing enzymatic cleavage of polyribonucleotides [128]. The hhRz consists of three helixes surrounding an evolutionary conserved catalytic core.…”

“…The hhRz consists of three helixes surrounding an evolutionary conserved catalytic core. This gives rise to an antisense complementary region that provides the unimolecular RNA the capacity to recognize and subsequently enzymatically cleave its target mRNA [128].…”

“…Both types of molecules (iRNA and hhRz) have been successfully used to degrade an incorrect RHO transcript responsible for dominant retinitis pigmentosa, a common subtype of IRD [128]. Another alternative is employing microRNAs (miRNAs), which act at the post-transcriptional level to regulate gene expression in the retina.…”